Platycodin D, a natural component of Platycodon grandiflorum, prevents both lysosome- and TMPRSS2-driven SARS-CoV-2 infection by hindering membrane fusion
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Abstract
An ongoing pandemic of coronavirus disease 2019 (COVID-19) is now the greatest threat to global public health. Herbal medicines and their derived natural products have drawn much attention in the treatment of COVID-19, but the detailed mechanisms by which natural products inhibit SARS-CoV-2 have not been elucidated. Here, we show that platycodin D (PD), a triterpenoid saponin abundant in Platycodon grandiflorum (PG), a dietary and medicinal herb commonly used in East Asia, effectively blocks the two main SARS-CoV-2 infection routes via lysosome- and transmembrane protease serine 2 (TMPRSS2)-driven entry. Mechanistically, PD prevents host entry of SARS-CoV-2 by redistributing membrane cholesterol to prevent membrane fusion, which can be reinstated by treatment with a PD-encapsulating agent. Furthermore, the inhibitory effects of PD are recapitulated by the pharmacological inhibition or gene silencing of NPC1 , which is mutated in patients with Niemann–Pick type C (NPC) displaying disrupted membrane cholesterol distribution. Finally, readily available local foods or herbal medicines containing PG root show similar inhibitory effects against SARS-CoV-2 infection. Our study proposes that PD is a potent natural product for preventing or treating COVID-19 and that briefly disrupting the distribution of membrane cholesterol is a potential novel therapeutic strategy for SARS-CoV-2 infection.
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SciScore for 10.1101/2020.12.22.423909: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources After blocking with 5% skim milk in TBST (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 20) for 1 h at room temperature (RT), the membranes were incubated in the in TBST at 4°C overnight with the following primary antibodies: rabbit anti-NPC1 (Novus, NB400-148), rabbit anti-NPC2 (Novus, NBP1-84012), rabbit anti-ACE2 (Abcam, ab15348), rabbit anti-TMPRSS2 (Abcam, ab92323), and mouse anti-GAPDH (Abcam, ab8245). anti-NPC1suggested: Noneanti-NPC2suggested: …SciScore for 10.1101/2020.12.22.423909: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources After blocking with 5% skim milk in TBST (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 20) for 1 h at room temperature (RT), the membranes were incubated in the in TBST at 4°C overnight with the following primary antibodies: rabbit anti-NPC1 (Novus, NB400-148), rabbit anti-NPC2 (Novus, NBP1-84012), rabbit anti-ACE2 (Abcam, ab15348), rabbit anti-TMPRSS2 (Abcam, ab92323), and mouse anti-GAPDH (Abcam, ab8245). anti-NPC1suggested: Noneanti-NPC2suggested: Noneanti-ACE2suggested: (Abcam Cat# ab15348, RRID:AB_301861)anti-TMPRSS2suggested: (Abcam Cat# ab92323, RRID:AB_10585592)anti-GAPDHsuggested: (Abcam Cat# ab8245, RRID:AB_2107448)Experimental Models: Cell Lines Sentences Resources Cell culture: H1299, A549, MRC-5, and CaCo2 cells were obtained Korean Cell Line Bank. A549suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)MRC-5suggested: ICLC Cat# HL95001, RRID:CVCL_0440)CaCo2suggested: CLS Cat# 300137/p1665_CaCo-2, RRID:CVCL_0025)In brief, HEK293T cells that reached 70-80% confluency in a 6-well plate were transfected with 1 μg of lentiviral backbone that contains expression cassettes for firefly luciferase, 0.75 μg of psPAX2 packing plasmid, and 0.5 μg of SARS-CoV-2 Spike plasmid (a gift from Fang Li, Addgene plasmid #145032) using Lipofectamine 3000 transfection reagent (Invitrogen, USA) following the manufacturer’s instructions. pMD2. HEK293Tsuggested: NoneFor experiments with drugs, H1299 cells were pre-treated for 1 h with each drugs before transduction. H1299suggested: NCI-DTP Cat# NCI-H1299, RRID:CVCL_0060)Vero cells were seeded at 1.2 × 104 cells per well and Calu-3 cells were seeded at 2.0 × 104 cells per well in black, 384-well, μClear plates (Greiner Bio-One, Austria), 24 h prior to the experiment. Calu-3suggested: NoneFor viral infection, plates were transferred into the BSL-3 containment facility and SARS-CoV-2 was added at a multiplicity of infection (MOI) of 0.0125 in Vero cells and 0.5 in Calu-3 cells. Verosuggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)Experimental Models: Organisms/Strains Sentences Resources Electrophysiology: For the brain acute slicing, 5 weeks C57BL/6J mice were anesthetized with isoflurane and decapitated. C57BL/6Jsuggested: RRID:IMSR_JAX:000664)Software and Algorithms Sentences Resources Molecular modelling of MβCD inclusion complexes: Schrodinger Maestro software 2017 suite (Maestro, Schrödinger, LLC, New York, NY) was used to prepare and score the MβCD binding complexes. Schrodinger Maestrosuggested: NoneFor sIPSC frequency and amplitude analyzing, Mini Analysis Program software (Synaptosoft) were used. Mini Analysis Programsuggested: (Mini Analysis Program, RRID:SCR_002184)9.0.0 was used (GraphPad Software). GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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